The long term goal of this project is to identify and understand the membrane-associated entities and processes contributing to the generation of a healthy ocular surface. This health depends on the proper proliferation and differentiation (acquisition or loss of cytosolic, cytoskeletal and membrane-associated components) by the cells of the limbo-corneal epithelial lineage. Earlier studies in corneal epithelial differentiation identified changes in the expression of cytokeratin and other markers in the transition to the terminally differentiated state. Very little is known, though, about the regulatory processes involved. In recent years we have shown that de novo (or enhanced) expression of gap junction (GJ) proteins (connexins; Cx) and gap junction-mediated intercellular communications (GJIC) are early events in both CE development and differentiation. Because the GJIC status is expected to determine the degree to which intracellular messengers are shared between cells of any compact cell population, changes in Cx expression could be critical mediators of the differentiation process, in particular its coordinated nature and/or proliferative changes. To examine these propositions, the current project will: I) Obtain detailed biochemical, structural and physiological information of the status and developmental dynamics of gap junctions in the mammalian limbo-corneal epithelium. II) Determine whether the absence of Cx43 (and GJIC) in the limbus reflects developmentally induced down-regulation of expression of the gene for this protein or changes in post-transcriptional/post-translational processing patterns that prevent establishment of gap junctions. III) Examine the effect of viral vector-inserted antisenses for the elimination of GJIC on proliferation and differentiation in primary cultures of limbal cells and, conversely, the effect of restitution GJIC function on the differentiation of GJIC-deficient immortalized CE rabbit cells.